Project Summary/Abstract Inwardly-rectifying K channel 4.1 (Kir4.1) is encoded by Kcnj10 and plays a dominant role in providing basolateral K conductance and in determining the negative membrane potential in the distal convoluted tubule (DCT). The critical role of Kir4.1 in the DCT is demonstrated by the report that loss-of-function mutations of Kcnj10 in the kidney cause tubulopathy, a disease which is reminiscent to Gitelman's syndrome including hypomagnesemia, hypokalemia and metabolic alkalosis. The renal phenotype of loss-function mutations of Kir4.1 observed in SeSAME/EAST syndrome is recapitulated in the kidney-specific Kir4.1 knockout (Ks-Kir4.1 KO) mouse model. We have demonstrated that Ks-Kir4.1 KO mice have severe hypokalemia and hypochloremic metabolic alkalosis, suggesting a defective membrane transport in the DCT. Kir4.1 interacts with Kir5.1 encoded by Kcnj16 to form a 40 pS K channel in the basolateral membrane of the DCT. While Kir4.1 is responsible for providing basolateral K conductance in the DCT, Kir5.1 may serve as a regulatory subunit for the heterotetramer. Kir5.1 contains a phosphor-threonine-based Nedd4 E3 ligase binding motif at its c-terminus (TPVT), which has been shown to be a binding site of Nedd4 in phosphatase CDC25c. The possibility that Kir5.1 may regulate Kir4.1 activity in the DCT through binding to Nedd4-2 is also suggested by our preliminary experiments showing that depletion of Kir5.1 and Nedd4-2 stimulates Kir4.1 activity which is known to stimulate NCC activity. NCC not only plays a role in mediating Na absorption in the DCT but also in regulating renal K excretion. Thus, the goal of the present application is to test hypothesis that Kir5.1 is the binding partner for Nedd4-2 E3 ligase which ubiquitinizes Kir4.1 and depolarizes membrane in the DCT and that Kir5.1 and Nedd4-2 E3 ligase play a key role in mediating the inhibitory effects of high Na and high K intake on the basolateral Kir4.1 and the apical NCC. The proposal has three specific aims: In specific aim 1, we will test whether Kir5.1(Kcnj16) is a binding partner of Nedd4-2 E3 ligase and that Nedd4-2 binding to Kir5.1 ubiquitinizes Kir4.1 and depolarizes the membrane in the DCT. In specific aim 2, we will test the hypothesis that Kir5.1 and Nedd4-2 E3 ligase are essential for mediating the inhibitory effect of high Na intake on Kir4.1 and NCC in the DCT. In specific aim 3, we will test the hypothesis that Kir5.1 and Nedd4-2 E3 ligase are essential for mediating the inhibitory effect of high K intake on Kir4.1 and NCC in the DCT. Since basolateral Kir4.1 plays a key role in regulating WNK-SPAK-NCC in the DCT , understanding the role of Kir5.1 and Nedd4-2 in regulating Kir4.1 will provide an integrated view regarding the regulation of K and Na transport in the distal tubules.